Authorship:
Finnur Freyr Eiriksson(1,2); Hrafnhildur Runolfsdottir(1); Vidar O. Edvardsson(3); Runolfur Palsson(1,3); Margrét Thorsteinsdóttir(1,2)
(1)University of Iceland, Reykjavik, Iceland (2)ArcticMass, Reykjavik, Iceland (3)Landspitali University Hospital, Reykjavik, Iceland
| Short Abstract Adenine phosphoribosyltransferase (APRT) deficiency results in excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA) causing kidney disease. Treatment with allopurinol and febuxostat prevents the disease in patients with APRT deficiency. Therapeutic drug monitoring is currently performed by urine microscopy but a more sensitive and reliable method is needed. A UPLC-MS/MS method was developed and utilized to measure the concentration of DHA in urine samples from 28 patients, before and after treatment with allopurinol or febuxostat. Significant changes were observed in urinary excretion of DHA after pharmacotherapy was initiated. A decrease in the DHA-to-creatinine ratio was observed with both allopurinol and febuxostat therapy. The developed UPLC-MS/MS assay will greatly facilitate clinical diagnosis and therapeutic drug monitoring in patients with APRT deficiency. |
Long Abstract
Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive error of adenine metabolism which results in excessive urinary excretion of poorly soluble 2,8-dihydroxyadenine (DHA). Patients with APRT deficiency have an excessive urinary excretion of poorly soluble DHA, causing crystalline nephropathy and chronic kidney disease. A significant number of patients are asymptomatic at diagnosis and end-stage kidney failure is not recognized until after kidney transplantation. Treatment with xanthine dehydrogenase (XDH) inhibitor allopurinol and febuxostat is known to prevent nephrolithiasis and chronic kidney disease in patients with APRT deficiency by reducing the synthesis of DHA. Therapeutic drug monitoring is currently done by urine microscopy where the absence of urinary DHA crystals has been considered indicative of adequate therapy. However, in some patients stone formation continues despite major decrease or even disappearance of urinary crystals while others remain free of stones. Therefore a more sensitive and reliable method to monitor treatment is needed. The purpose of this work was to develop a more reliable assay based on quantification of DHA and adenine in human urine by UPLC-MS/MS.
The quantification method for DHA and adenine was developed and optimized utilizing a chemometric approach. Experimental screening of variables was performed in negative and positive ionization mode. Six quantitative and qualitative variables were optimized by D-optimal design and related to sensitivity and retention time utilizing PLS regression. Pre-treatment handling of urine samples were tested prior to analysis of patients samples before and after treatment with Allopurinol or Febuxostat. The optimized UPLC-MS/MS method was used to measure the concentration of DHA in 24 hr and random urine samples from 28 patients with APRT deficiency, before and after treatment with allopurinol or the alternative agent febuxostat. Patients with eGFR >60 ml/min/1.73 m2 listed in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, currently receiving allopurinol therapy, were enrolled in a 42-day pilot study. After 7 day washout period, the subjects were prescribed 400 mg of allopurinol in a single daily dose for 14 days. After a second 7 day washout period, all subjects were prescribed 80 mg febuxostat in a single daily dose for another 14 days. Twenty-four hour urinary DHA excretion was evaluated at the end of the first washout period and at the end of allopurinol and febuxostat treatment periods (days 7, 21 and 42). Urinary DHA was measured using UPLC-MS/MS and expressed as DHA-to-creatinine ratio (ng/mmol).
A reliable UPLC-MS/MS assay for simultaneous quantification of urinary DHA and adenine for diagnosis and monitoring of pharmacotherapy in patients with inborn errors of purine metabolism was developed by chemometric approach. This approach required only a fraction of the number of experiments that would have been required by changing one separate factor at time (COST) approach. The recovery was significantly increased by diluting the urine samples with 10mM NH4OH prior to analysis. Preliminary data suggest that it is very important to standardize pre-treatment; sample handling, aliquoting and storage conditions of urine samples prior to quantification of DHA and adenine by the UPLC-MS/MS assay. Significant changes were observed in urinary excretion of DHA and adenine after pharmacotherapy was initiated. To date, 6 patients of 10 have completed their participation in the study. The median (range) urinary DHA-to-creatinine ratio was 3207 (1620-5374) ng/mmol off therapy, 2105 (813-4014) ng/mmol on allopurinol therapy and 422 (141-446) ng/mmol on febuxostat treatment. Statistical significance was not calculated due to the small number of subjects. A decrease in the DHA-to-creatinine ratio was observed with both allopurinol and febuxostat therapy. In the prescribed doses, febuxostat appears to be more effecacious than allopurinol in reducing DHA excretion in patients with APRT deficiency. These results need to be confirmed in a larger patient sample and the quantification method needs to be validated. We belive that the developed UPLC-MS/MS assay will greatly facilitate clinical diagnosis and therapeutic drug monitoring in patients with APRT deficiency.